KR101444877B1 - In situ crosslinking hydrogel comprising γ-polyglutamic acid and method for producing the same - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a biodegradable and biocompatible hydrogel which can be used as a sealant for inhibiting leakage of blood or air during surgical operation, a tissue adhesive, an adhesion inhibitor, a drug delivery carrier, etc., During the operation, the solution is kept in the aqueous solution for a certain period of time, so that the solution is not required to be used immediately. The gel is formed immediately from the wound site, and the adhesive force to the biotissue is excellent, so that leakage of blood or air is suppressed, and it is decomposed and absorbed or discharged in the living body, and there is no toxicity to the living body. The gelation time can also be adjusted and controlled to a desired degree. Therefore, the hydrogel can be used preferably for various purposes such as biotissue bonding and the like.

Description

In situ crosslinking hydrogel comprising γ-polyglutamic acid and method for producing the same,

TECHNICAL FIELD The present invention relates to a biodegradable and biocompatible hydrogel which can be used as a sealant, a tissue adhesive, an adhesion inhibitor, a drug delivery carrier, and the like, which inhibits leakage of blood or air during surgical operation, and a method for producing the same.

Fibrin glue and protein adhesives have been mainly used for medical use, particularly for surgical surgical sealants and adhesives. However, fibrin glue has a disadvantage that it is easily detached from the surgical site due to its low adhesive strength, and there is a fear of disease transmission such as viral infection because it is a blood product. Examples of the protein adhesives include products using a mixture of albumin (Bovine serum albumin) and glutaraldehyde (USP 5,385,606, product name: bioglue?), Gelatin, resorcinol and formaldehyde : GRF glue?) Are used but all have drawbacks in that biocompatibility is deteriorated due to concerns of infection due to use of animal-derived proteins and toxicity of aldehyde used as a crosslinking agent.

Recently, studies on the in situ forming hydrogels using synthetic polymers (mainly polyethylene glycol (PEG)) or carbohydrates (mainly dextran) have been actively conducted as substitutes for blood products and protein materials, to be. Depending on the kind of crosslinking reaction, it can be classified into radical polymerization reaction and substitution reaction between nucleophile and electropile. The substitution reaction can be further divided into imine bond and amide bond depending on the type of bond. As an example of radical polymerization, FocalSeal? Photo-activated polyethylene glycol (PEG), which is marketed under the trade name of Genzyme, has been reported to exhibit higher strength than fibrin sealants, but a light source and photoinitiator are required for cross-linking reactions, And there is a disadvantage in use. The imine bond is formed by the reaction of an aldehyde group and an amine group. The aldehyde dextran is used as the electrophile, and chitosan or aminated polyvinyl alcohol (US2005 / 00028930), multi amino PEG (US2006 / 0078536, Biomaterials 29 2008) 4584-4591) and the use of ε-polylysine synthesized through microbial culture (WO2006 / 080523) has been reported. However, in the case of aldehyde dextran, the problem of the toxicity of the aldehyde group and the lack of stability, which is easily oxidized to lose the reactivity, remains. Amide bond is activated by reacting a carboxyl group with a succinimidyl ester group to react with an amine group such as a nucleophile. (US 5,162,430) and serum albumin (US RE 38,827) are used as the nucleophiles, but as described above, there is still a concern that the transfer of the disease Remains. In addition, a hydration gel using a crosslinking reaction (US 6,602,952) with chitosan or methoxy PEG-conjugated chitosan has been disclosed, but in this invention, the gelation time is not suitable for on-site crosslinked hydrogel in about 2 hours. As the synthetic nucleophiles, multi-amino- or multi-mercapto-PEG (US 5,874,500, product name CoSeal?) And trilysine (US 6,566,406, product name DuraSeal?) Have been developed and commercialized. As described above, many examples using polyethyleneglycol having excellent biocompatibility as synthetic polymers have been reported.

γ-polyglutamic acid is a water-soluble, anionic, biodegradable, and biocompatible polymer biosynthesized by Bacillus subtilis, a soybean fermented food microorganism, which is a γ-polypeptide in which γ-carboxyl group and α-amino group of glutamic acid are amide bonded. Attempts have been made to cross-link this? -Polyglutamic acid to be used as an absorbent or a medical hydrogel, and a representative example thereof is as follows.

Japanese Patent Application Laid-Open No. 1999-276572 discloses a method of ion-binding a polygamatoglutamic acid complex prepared by hydrogen bonding of a quaternary amine salt such as chitosan to a carboxy anion of a? -Polyglutamate to a surgical suture, a wound dressing, Prevention material, hemostatic material, and the like.

Crosslinking by chemical bonding using a crosslinking reagent, and Japanese Patent Laid-Open Publication No. 1999-343339 discloses a crosslinking reaction by adding a polyepoxy compound such as diethyleneglycol diglycidyl ether to? -Polyglutamic acid with a crosslinking agent And it is disclosed in WO 2007/132785 that it is applied as an anti-adhesion agent. However, it is impossible to apply it for the purpose of in situ gelation in a medical field at a reaction condition of 40 ° C * 48 hours or 90 ° C * 30 minutes.

An example using water soluble carbodiimide as a condensing agent for promoting the reaction between the carboxyl group of γ-polyglutamic acid and the nucleophile has been reported. An example of using 3- (3-dimethyl aminopropyl) -1-ethyl carbodiimide (EDC) as a water-soluble carbodiimide using biodegradable fructose, lysine or chitosan as a double crosslinking compound is disclosed in Japanese Patent Laid- 128899. However, this reaction condition is also very long at room temperature for 24 hours. And J. Appl. Polym. Sci. 65, pp. 1889-1896, 1997, a γ-PGA-EDC precipitate is prepared through the reaction of γ-polyglutamic acid and EDC, and 1,3-propane diamine is added as a crosslinking agent to prepare a hydrated gel. However, even under this condition, standing for 1 day after mixing is described, and the yield is also very low, below 10%.

It has been disclosed that gelatin is formed within 30 seconds upon reaction with gelatin using succinimide -la-polyglutamic acid as a crosslinking agent in Japanese Patent Laid-Open Publication No. 1997-103479 and Biomaterial 19 (1998) 1869-1876, The gelatinization reaction of the gelatin which is naturally gelled at a temperature below the temperature has helped, and the? -Polyglutamic acid itself did not become the main component of the hydrogel.

Accordingly, there is a continuing demand for a hydrogel which is short in gel time, exhibits biocompatibility and biodegradability, has excellent adhesive strength and rupture strength, and is preferably applicable to tissue adhesion and the like.

Polyglutamic acid derivative having improved activity in an aqueous solution and biodegradable and biocompatible γ-polyglutamic acid derivative having a tissue adhesive force necessary for medical tissue sealant and having biodegradability It is an object of the present invention to provide a hydrogel.

The present invention also provides a method for producing the γ-polyglutamic acid derivative and the hydrogel, a kit for preparing the hydrogel, and a tissue adhesive composition comprising the component capable of forming the hydrogel.

In order to maintain the activity for a predetermined time in an aqueous solution of each substance while satisfying the conditions for forming a hydrogel, the inventors of the present invention have found that a biosynthetic polyamino acid capable of mass production by fermentation of microorganisms retains a carboxyl group in its side chain, Polyglutamic acid derivative activated with at least a part of the amino group or thiol group is widely used as a biomaterial and a polyethylene glycol polymer having an amino group or a thiol group on the side chain is used to have a tissue adhesive force necessary as a medical tissue sealant, It is possible to produce a hydrogel having biodegradability and biocompatibility, thereby completing the present invention. In particular, the activated γ-polyglutamic acid derivative is characterized in that an alkyl group is introduced into γ-polyglutamic acid in order to improve degradation of activity in an aqueous solution, which is a weak point of γ-polyglutamic acid activated by a conventional succinimide group.

Accordingly, one embodiment of the present invention provides a γ-polyglutamic acid derivative activated by introduction of an alkyl group and a succinimide in at least a part of carboxyl groups.

Another embodiment of the present invention is a method for producing γ-polyglutamic acid-alkanolamine by reacting at least a part of carboxyl groups of γ-polyglutamic acid with a lower alkanolamine having 1 to 5 carbon atoms to form γ-polyglutamic acid-alkanolamine; Wherein the hydroxy group of the? -Polyglutamic acid-alkanolamine is selected from the group consisting of an anhydride of an acid selected from the group consisting of glutaric acid and succinic acid, or an anhydride of an acid selected from the group consisting of 1-halovaleric acid, 1-halopropionic acid and 1-halomethylcarboxylic acid A second step of reacting with 1-haloalkanoic acid to form a carboxy terminal introduced with an alkyl group; And a third step of reacting the formed carboxyl terminal with N-hydroxysuccinimide or N-hydroxysulfosuccinimide to form at least a carboxyl group-activated? -Polyglutamic acid derivative. Polyglutamic acid derivative according to one embodiment of the present invention.

Another embodiment of the present invention provides a hydrogel comprising a cross-linked product of a γ-polyglutamic acid derivative according to an embodiment of the present invention and a polyethylene glycol-based polymer having a plurality of nucleophilic functional groups cross-linked.

In another embodiment of the present invention, there is provided a method for producing γ-polyglutamic acid-alkanolamine by reacting at least a part of carboxyl groups of γ-polyglutamic acid with a lower alkanolamine having 1 to 5 carbon atoms to form γ-polyglutamic acid-alkanolamine; Wherein the hydroxy group of the? -Polyglutamic acid-alkanolamine is selected from the group consisting of an anhydride of an acid selected from the group consisting of glutaric acid and succinic acid, or an anhydride of an acid selected from the group consisting of 1-halovaleric acid, 1-halopropionic acid and 1-halomethylcarboxylic acid A second step of reacting with 1-haloalkanoic acid to form a carboxy terminal introduced with an alkyl group; A third step of reacting the formed carboxyl terminal with N-hydroxysuccinimide or N-hydroxysulfosuccinimide to form at least a carboxyl group-activated? -Polyglutamic acid derivative; And a fourth step of cross-linking the activated γ-polyglutamic acid derivative with a polyethylene glycol-based polymer having a plurality of nucleophilic functional groups.

According to another embodiment of the present invention, there is provided a kit for preparing a hydrogel, and a tissue adhesive composition comprising a γ-polyglutamic acid derivative according to an embodiment of the present invention and a polyethylene glycol-based polymer having a plurality of nucleophilic functional groups .

Hereinafter, the? -Polyglutamic acid derivative, the hydrogel, the method for producing the same, and the tissue adhesive according to embodiments of the present invention will be described in detail.

In order to produce an adhesive water-soluble gel, crosslinking takes place by bonding between mutually-reactive polymer chains having at least two functional groups, and at least one of the polymers preferably forms a covalent bond with the surface of the tissue simultaneously with the crosslinking reaction.

The chemical covalent bond is formed by a reaction between an electrophile and a necleophile. The electrophilic functional group is an amino group (-NH ₂ ), a thiol group (-SH), or a hydroxyl group (-OH). ≪ / RTI > Such an electrophilic functional group may be an aldehyde group and an activated ester group. For example, when an amino group is reacted with the amino group, a crosslinking reaction and a tissue adhesion occur simultaneously through an imine bond and an amide bond. When at least a part of the? -Polyglutamic acid is reacted with a specific compound to convert it into a succinimide ester group or the like and activated, the? -Polyglutamic acid thus activated is a polyethylene glycol group having a nucleophilic functional group such as an amine group, a thiol group or a hydroxyl group It has been found that a gel can be formed by causing a rapid gelation (crosslinking reaction) with a polymer even at room temperature. Therefore, the hydrogel is formed in a gel state immediately in a medical field, and is suitably applied to tissue adhesion and the like. Further, the activated γ-polyglutamic acid derivative according to a preferred embodiment of the present invention may be prepared by introducing an alkyl group into γ-polyglutamic acid in order to improve the degradation of the γ-polyglutamic acid in the aqueous solution, which is a weak point of? -Polyglutamic acid activated by the existing succinimide group .

Thus, the activated γ-polyglutamic acid derivative into which the alkyl group is introduced can form a gel with a polyethylene glycol-based polymer having a nucleophilic functional group such as an amine group, a thiol group or a hydroxyl group by causing rapid gelation at room temperature. In addition, the activated γ-polyglutamic acid derivatives have improved stability in aqueous solution without the use of alkyl groups immediately after dissolution, maintaining an initial level of gelation time, burst strength and adhesion strength for more than 2 hours after dissolution. The hydrogel having such properties can be adhered to tissues with excellent adhesive strength, and can be applied to a biotissue in a very preferable manner, and thus can be effectively used as a medical tissue sealant or the like.

The γ-polyglutamic acid derivative and the hydrogel according to this embodiment will be described in more detail as follows.

The term "hydrogel" as used herein refers to a polymer matrix capable of swelling, and may have a crosslinked structure including covalent bonds or noncovalent bonds. In addition, the hydrogel may include a three-dimensional network structure of the crosslinked structure, and may absorb water to form an elastic gel.

The hydrogel according to one embodiment of the present invention comprises a crosslinked product in which at least a carboxyl group-activated γ-polyglutamic acid derivative and a polyethyleneglycol-based polymer having a plurality of nucleophilic functional groups are crosslinked, and the γ-polyglutamic acid The derivative is obtained by introducing a lower alkanolamine having 1 to 5 carbon atoms in at least a part of the carboxyl group as a linker and reacting the hydroxy terminal of the introduced linker with a cyclic alkyl anhydride to form a carboxyl group at the terminal, The succinimide ester may be activated by succinimide, resulting in introduction of a succinimide ester derivative containing an alkyl group.

The lower alkanolamine having 1 to 5 carbon atoms used as the linker is, for example, aminomethanol, 1-amino-2-propanol, 1-amino- 3-propanol, 1-amino-4-butanol, 1-amino-5-pentanol or monoethanolamine (MEA or 1-amino -2-ethanol), preferably monoethanolamine.

The succinimide ester derivatives are, depending on the type of the introduction of an alkyl group, a succinimidyl valeric acid _{(Succinimidyl Valerate, SVA: -CH 2} CH 2 CH 2 CH 2 -CO-NHS), succinimidyl-glutaric acid (Succinimidyl Glutarate, SG:: -CO-CH 2 CH 2 CH 2 -CO-NHS), succinimidyl propionate _{(Succinimdyl propionate, SPA: -CH 2} CH 2 -CO-NHS), succinimidyl succinate (succinimidyl succinate , SS: -CO-CH ₂ CH ₂ -CO-NHS), succinimidyl carboxymethylated (SCM: -CH ₂ -CO-NHS), and the like.

The γ-polyglutamic acid derivative may be represented by the following general formula (1).

In Formula 1,

The sum of l, m and n is an integer from 390 to 15,500,

Wherein the ratio of 1, m and n is 1: m: n = 0 to 0.5: 0.2 to 0.5: 0.2 to 0.8,

L is a linker, M is independently H, an alkali metal or an alkaline earth metal, R is CH ₂ ,

b is 0 or 1, and c is an integer of 1 to 5.

The linker is preferably -HN- (R) a-O-, wherein R in the linker is CH2 and a is an integer of 1 to 5.

The - (CO) b- (R) c-CO- of the above formula (1) is preferably -CH ₂ CH ₂ CH ₂ CH ₂ -CO-, -CO-CH ₂ CH ₂ CH ₂ -CO-, -CH ₂ CH ₂ -CO-, -CO-CH ₂ CH ₂ -CO- or -CH ₂ -CO-.

The hydrogel can be rapidly gelated in an aqueous solution at pH 7.2 to 11.0 at 37 ° C, which corresponds to the physiological condition of the hydrogel, and in situ gel formation can take place on living organisms or organ or tissues of the human body . Under the physiological conditions as described above, the crosslinking reaction time until gelation can be made within 10 minutes, more preferably within 2 minutes, and still more preferably within 30 seconds. Therefore, the hydrogel is formed in a gel state immediately at a medical field and applied to a living tissue, so that it can be effectively used for tissue adhesion.

The molar ratio of the ester group of the activated γ-polyglutamic acid derivative in the carboxyl group of the γ-polyglutamic acid derivative may preferably be 0.10 to 0.99, more preferably 0.30 to 0.80, and most preferably 0.5 to 0.7. By activating at such a molar ratio, the gelation time of the hydrogel can be shortened and the adhesion and strength to the living tissue can be optimized. The higher the degree of activation, the greater the number of crosslinking points forming the three-dimensional network structure of the hydrogel formed by the crosslinking reaction, and the physical strength such as the compressive strength and the tensile strength of the hydrogel also increases, and the bonding strength of the tissue increases.

The? -Polyglutamic acid before activation can be represented by the following general formula (1).

≪ General Formula 1 &

In the above,

n is from 390 to 15,500, preferably from 3,900 to 7,800,

M is H, an alkali metal or an alkaline earth metal (e.g., Na, K, Ca or Mg, preferably Na).

The weight average molecular weight of the? -Polyglutamic acid may be preferably 50,000 to 2,000,000 daltons, more preferably 50,000 to 1,000,000 daltons.

If the molecular weight is too high, the solubility of the synthesized activated γ-polyglutamic acid derivative tends to be low and it takes a lot of time to dissolve.

The polyethylene glycol-based polymer, which is a polymer forming a crosslinked product with the activated? -Polyglutamic acid derivative according to a preferred embodiment of the present invention, may preferably have an amine group or a thiol group bonded to the terminal. For example, such a polyethylene glycol-based polymer has a structure in which a polyethylene glycol repeating unit is bonded to each hydroxy group of a polyhydric alcohol having a valence of 2 or more, preferably 2 to 12, and an amine group, a thiol group or a hydroxy group is bonded to the terminal Lt; / RTI > More specifically, such a polyethylene glycol-based polymer may be represented by the general formula (2).

In the above formula (2), I is a radical derived from a polyhydric alcohol having 2 to 12 valences, in which hydrogen is replaced with - (CH ₂ CH ₂ O) n -CH ₂ CH ₂ X in each hydroxy group of the polyhydric alcohol, X is an amine group, a thiol group or a hydroxyl group, n is 19 to 170, and m is an integer of 2 to 12, which is the same as the number of hydroxyl groups of the polyhydric alcohol derived from I above.

In Formula 2, specific examples of I include diols such as ethylene glycol, propanediol, butanediol, pentanediol, and hexanediol; Or glycerol, erythritol, threitol, pentaerythritol, xy1itol, adonitol, sorbitol, mannitol, palatinose, a radical derived from a 3-12-fold polyol selected from trisaccharides such as palatinose, maltose monohydrate or maltitol, or trisaccharides such as D-raffinose pentahydrate, . More specifically, the I may be a radical derived from a polyhydric alcohol having 4 to 12 valences.

Examples of the polyethylene glycol-based polymer belonging to the category of the formula (2) include a polymer in which a polyethylene glycol repeating unit having a terminal nucleophilic functional group is bonded to a radical derived from pentaerythritol or sorbitol, for example, Or 4.

In the above formulas (3) and (4), X and n are as defined in the above formula (2).

Such a polyethylene glycol-based polymer may form an amide bond, thioamide bond or ester bond with the activated γ-polyglutamic acid derivative of the above formula (1) by including many nucleophilic functional groups such as an amine group, a thiol group or a hydroxyl group And a crosslinked structure is formed therefrom to form a crosslinked body and a hydrogel having a three-dimensional network structure. In particular, such a polyethylene glycol-based polymer causes a rapid crosslinking reaction with the activated γ-polyglutamic acid derivative, thereby making it possible to provide a hydrogel having a short gel time.

Such a polyethylene glycol-based polymer is a typical biocompatible polymer among synthetic polymers, and a hydrogel containing the crosslinked body of the polyethylene glycol-based polymer may exhibit biocompatibility suitable for tissue adhesion and the like. The polyethylene glycol-based polymer may be produced by a conventional method such as addition polymerization of ethylene oxide to a polyhydric alcohol and introduction of a nucleophilic functional group.

The weight average molecular weight of the polyethylene glycol-based polymer may be 5,000 to 30,000 daltons, specifically 10,000 to 20,000 daltons. If the weight average molecular weight is too small, the gelation time for forming the crosslinked product and the hydrogel may become long, or the gelation reaction may not take place. On the contrary, if the molecular weight of the polyethylene glycol polymer is excessively large, The property may be degraded.

Further, in the hydrogel of this embodiment, the nucleophilic functional group of the polyethylene glycol-based polymer is 0.1 to 2.0, specifically 0.2 to 2, 1.0, more specifically 0.4 to 0.6. By satisfying such a molar ratio, the hydrogel can have an excellent adhesive strength and rupture strength to a living body tissue, and crosslinking of a hydrogel can also be optimized. However, if the molar ratio is too low, the strength of the hydrogel may be weakened due to a small number of crosslinking points.

According to another embodiment of the present invention, there is provided a process for producing the above-mentioned γ-polyglutamic acid derivative and a hydrogel in which the γ-polyglutamic acid derivative and the polyethylene glycol-based polymer are crosslinked.

Preferably, the preparation method comprises a first step of reacting at least a part of carboxyl groups of? -Polyglutamic acid with a lower alkanolamine having 1 to 5 carbon atoms to form? -Polyglutamic acid-alkanolamine; Wherein the hydroxy group of the? -Polyglutamic acid-alkanolamine is selected from the group consisting of an anhydride of an acid selected from the group consisting of glutaric acid and succinic acid, or an anhydride of an acid selected from the group consisting of 1-halovaleric acid, 1-halopropionic acid and 1-halomethylcarboxylic acid A second step of reacting with 1-haloalkanoic acid to form a carboxy terminal introduced with an alkyl group; And a third step of reacting the formed carboxyl terminal with N-hydroxysuccinimide or N-hydroxysulfosuccinimide to form at least a part of the γ-polyglutamic acid derivative activated with a carboxyl group.

The first to third steps of forming the at least a carboxyl group-activated? -Polyglutamic acid derivative will be described with reference to the following reaction formula (1). The following Reaction Scheme 1 shows an example of a method for producing a? -Polyglutamic acid derivative according to an embodiment of the present invention, wherein monoethanolamine (MEA) is used as a linker, and an anhydride of succinic acid is used to form a carboxy terminal And then activated using NHS.

[Reaction Scheme 1]

Preferably, all of the reactions in the above 1 to 3 stages are carried out at 0 to 150 ° C, specifically 20 to 70 ° C, and the reaction time is 0.5 to 35 hours, preferably 5 to 24 hours.

The step 1 is a step of introducing a linker to the carboxyl group of? -Polyglutamic acid, wherein at least a part of the? -Polyglutamic acid carboxy group is reacted with a lower alkanolamine having 1 to 5 carbon atoms to form? -Polyglutamic acid-alkanolamine The linker can be introduced.

The lower alkanolamine having 1 to 5 carbon atoms is preferably selected from the group consisting of aminomethanol, 1-amino-2-propanol, 1-amino-3-propanol, 1-amino-4-butanol, MEA or 1-amino-2-ethanol), preferably monoethanolamine.

Preferably, the alkanolamine may be reacted in the presence of a carbodiimide compound such as DCC and N-hydroxysuccinimide (or N-hydroxysulfosuccinimide), preferably the carbodiimide compound And the N-hydroxysuccinimide (or N-hydroxysulfosuccinimide) and the alkanolamine are added to the molar unit of the carboxyl group contained in the? -Polyglutamic acid prior to the activation, respectively, in the range of 0.1 to 2, Can be reacted at a molar ratio of 1 to 2.

Preferably, step 1 may be carried out in an aprotic solvent such as DMSO (Dimethyl sulfoxide), DMF (Dimethyl formamide), and Formamide. The quantum solvent is undesirable because it can react with the activated NHS-ester, and other common organic solvents are not preferred because the reactants are insoluble.

The step 2 is a step of introducing a carboxyl group into which an alkyl group has been introduced. The step 2 is a step of introducing an alkyl group-introduced carboxyl group into an amphoteric organic solvent such as formamide, dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) Can be reacted with an anhydride of an acid selected from the group consisting of glutaric acid and succinic acid to form a carboxy terminal, and the carboxyl group can be formed by reacting the carboxylic acid with a carboxylic acid selected from the group consisting of 1-halovaleric acid, 1-halopropionic acid, The carboxyl terminal may be formed by reacting with 1-halo alkanoic acid. .

Preferably, the anhydride or 1-haloalkanoic acid of the acid is used in a molar ratio of 3 to 8, more preferably 4 to 6, to the molar unit of the lower alkanolamine introduced to form the? -Polyglutamic acid-alkanolamine And the carboxyl group can be introduced into the? -Polyglutamic acid-alkanolamine at 100 mol%.

The step 3 is a step of forming an activated γ-polyglutamic acid derivative, wherein the carboxyl terminal formed through the above two steps is reacted with N-hydroxysuccinimide or N-hydroxysulfosuccinimide to form at least a part of the carboxyl group Polyglutamic acid derivative of the formula (1) can be formed.

The above three steps can preferably proceed in the presence of a carbodiimide based compound such as, for example, dicyclohexylcarbodiimide (DCC), whereby the reaction step can proceed more efficiently.

Preferably, the carbodiimide compound is added to the molar unit of the carboxyl group formed through the reaction of the acid with an anhydride or 1-haloalkanoic acid in a molar ratio of 0.1 to 3, more preferably 1 to 2, N-hydroxysuccinimide or N-hydroxysulfosuccinimide at a molar ratio of 0.1 to 3, more preferably 1 to 2. The reaction may be carried out in the presence of a base.

The molar ratio of the ester group of the? -Polyglutamic acid derivative activated through step 3 above is preferably 0.10 to 0.99, more preferably 0.30 to 0.80, and most preferably 0.5 to 0.80, relative to the carboxyl group of? -Polyglutamic acid before activation. 0.7. By activating at such a molar ratio, the gelation time of the hydrogel can be shortened and the adhesion and strength to the living tissue can be optimized.

On the other hand, after the γ-polyglutamic acid derivative activated by the above-mentioned method is prepared, a step of cross-linking the γ-polyglutamic acid derivative with a plurality of nucleophilic functional group-having polyethylene glycol-based polymers may be further performed to prepare a crosslinked product and a hydrogel.

According to a preferred embodiment of the present invention, there is provided a method for producing a hydrogel, comprising the step of cross-linking a γ-polyglutamic acid derivative of Formula 1 with a polyethylene glycol-based polymer having a plurality of nucleophilic functional groups. According to this preparation method, a hydrogel having a crosslinked structure can be obtained even in a physiological condition near room temperature. In particular, even in a medical field, such a hydrogel can be immediately applied to facilitate tissue adhesion and the like.

Specifically, after the first to third steps of preparing the γ-polyglutamic acid derivative, the method for preparing the hydrogel includes reacting the activated γ-polyglutamic acid derivative with a polyethyleneglycol-based polymer having a plurality of nucleophilic functional groups And a fourth step of performing a cross-linking reaction.

The four-step crosslinking reaction can be carried out in a solution state in which an activated γ-polyglutamic acid derivative and a polyethylene glycol-based polymer are mixed. For example, the first solution containing the solution of the activated γ-polyglutamic acid derivative and the second solution containing the solution of the polyethylene glycol-based polymer may be mixed.

At this time, in order to obtain a homogeneous gel in the mixing of the first liquid and the second liquid, it is preferable to mix them with a solution of appropriate concentration. The crosslinking density can be controlled by the concentration of the polymer. The higher the concentration of the polymer, the higher the degree of crosslinking. However, in the case of one solution in which an activated γ-polyglutamic acid derivative (over 1000 K) having a larger molecular weight than the polyethylene glycol polymer is dissolved, the solubility decreases below a certain concentration, So that uniform mixing is difficult. In addition, precipitation occurs during mixing and does not produce a uniform hydrogel. If the concentration of the polymer is too low, the degree of crosslinking is low and the strength of the gel is weak or gel is not produced.

Therefore, the concentration of the polymer, that is, the total concentration of the activated γ-polyglutamic acid derivative and the polyethylene glycol-based polymer relative to the total solution is preferably 1 to 20% by weight, more preferably 5 to 15% 10% by weight.

Also, the concentration ratio between the activated γ-polyglutamic acid derivative and the poly (ω) -ylene-based polymer may be 5 to 10:10 to 15 (wt%).

As a solvent for preparing the first solution and the second solution, distilled water and a buffer solution such as physiological saline, sodium hydrogencarbonate (NaHCO ₃ ), boric acid, phosphoric acid and the like may be used without toxicity.

The buffer in the solvent affects the gelation time. That is, depending on the type of the buffer, the gelation reaction may or may not occur, and the gelation time may be adjusted quickly or slowly. The buffer should be made using a salt having a pKa similar to the pH of the solid component of the first and second liquids, and then the buffering effect is maximized to help reduce the deactivation of the activated gamma-polyglutamate derivative in aqueous solution You can give.

Preferably, a sodium phosphate buffer can be used as a buffer solution to prepare the first liquid. Also preferably, a mixed buffer of sodium phosphate and sodium carbonate can be used to prepare the second solution. The mixing buffer may preferably be a mixture of sodium phosphate and sodium carbonate in a volume ratio of 1: 9 to 9: 1.

The buffer solution of the first solution is preferably used in a concentration of 0.01 to 0.3 mol, more preferably 0.05 to 0.1 mol, and the buffer solution of the second solution is preferably used in an amount of 0.01 to 0.5 mol, Can be used in a concentration of 0.1 to 0.3 mol.

The solid component of the first solution and the second solution can be easily sterilized by radiation sterilization, preferably sterilized by irradiation with gamma rays of 10 to 50 kGy, more preferably gamma rays of 20 to 30 kGy do. Such a sterilization treatment can be carried out by setting conditions so as not to adversely affect the properties of the curing time and other hydrogels.

When the first solution and the second solution are mixed, an amide, thioamide or ester bond is formed between the activated carboxyl group (e.g., succinimide ester group) of the? -Polyglutamic acid derivative and the nucleophilic functional group, A hydrogel having a dimensional network structure can be formed. As a result, the crosslinking reaction can proceed preferably at 0 to 50 캜, more preferably at 25 to 40 캜, and from 1 second to 200 seconds, preferably from 2 seconds to 100 seconds, more preferably from 3 seconds (Gelation) can be initiated within a period of from 30 seconds to 50 seconds.

The preferable time from mixing to gelation may be somewhat different depending on the application, but preferably is within 10 minutes, more preferably within 2 minutes, and more preferably within 30 seconds.

However, when the gelation time is less than 2 seconds, injection needle or spray tip may become clogged and it may be difficult to apply smoothly. Also, because there is not enough time to mix each component, uneven gel is formed or covalent bond with the tissue surface is difficult The adhesive strength may be lowered. On the other hand, if the gelation time is too long, it may flow into the solution at the application site before the gel is formed, which is not easy to use. Therefore, it is preferable that the time is 3 seconds or more, especially 5 seconds or more and 15 seconds or less, until penetration into the living tissue and maintaining the high adhesive force and burst strength is achieved.

The hydrogel as described above has excellent properties through rapid gelation even under physiological conditions, so that it can be very suitably applied for tissue adhesion in a medical field. The application may be performed, for example, using a mechanism such as a double barrel syringe, but is not limited thereto.

The hydrogel prepared by the above method may be lyophilized to provide a sponge or a sheet form, or may be pulverized. In this form, the hydrogel may be used as an adhesion inhibitor, an absorbent, a drug delivery device, or the like. Also, as will be described in more detail below, a kit or a composition containing each component (the activated? -Polyglutamic acid derivative of Chemical Formula 1 and a polyethylene glycol-based polymer) for forming a hydrogel is subjected to a cross- So that it can be applied to adhesion of living tissue. For application to such tissue adhesion, a mixture containing an activated γ-polyglutamic acid derivative and a polyethylene glycol-based polymer, for example, a mixed aqueous solution, is formed on the living tissue, and then the aqueous solution is crosslinked to form a hydrogel A coating can be formed on the living tissue.

According to another embodiment of the present invention, there is provided a kit for the production of the hydrogel described above. Such a kit for preparing a hydrogel may include a? -Polyglutamic acid derivative of Formula (1) wherein at least a part of carboxyl groups are activated and a polyethylene glycol polymer having a plurality of nucleophilic functional groups, as described above.

As noted above, the hydrogel of one embodiment can be obtained at a rapid gelation rate under physiological conditions. Therefore, such a hydrogel can be used for tissue adhesion or the like, in the form of a kit or a composition before gelation containing each component for its formation. For example, after a kit or a composition containing each of the above components is applied to a living tissue, gelation is made on the living tissue to form a hydrogel, and the function of tissue adhesion can be shown. For such purposes and the like, the kit of another embodiment described above may be used.

According to a specific embodiment, such a kit for preparing a hydrogel may comprise a first liquid comprising an aqueous solution of activated? -Polyglutamic acid derivative and a second liquid comprising an aqueous solution of a polymer having nucleophilic functional groups. These kits can be mixed with aqueous solutions of the first solution and the second solution. In particular, it is possible to mix the first solution and the second solution to form gels in situ (in-situ forming hydrogel) .

When the two-component reactive sealant is used as described above, the mixing and use of the first solution and the second solution can be carried out by various methods. For example, it is possible to apply one of the stock solutions of the first and second liquids to the surface of the adherend and subsequently to apply the other solution, or the first liquid and the second liquid may be mixed in a double-barrel syringe a barrel syringe) may be mixed and applied. In some cases, in addition to the use as a sealant, a sheet made of a gel resin or the like may be used for the purpose of preventing adhesion. The mixing ratio (volume ratio) of the first liquid and the second liquid is usually set to 0.5 to 2.0 (the ratio of the second liquid to the first liquid, and vice versa).

On the other hand, according to another embodiment of the present invention, a tissue adhesive composition using the above-mentioned hydrogel is provided. As described above, the hydrogel is in the form of a composition before gelation containing each component for its formation, and can be preferably used for biotissue bonding. Thus, one embodiment of the tissue adhesive composition comprises a component for the production of a hydrogel, for example, a γ-polyglutamic acid derivative of Formula 1 wherein at least some carboxyl groups are activated, and a polyethylene glycol-based And may include a polymer.

The composition of an embodiment comprising at least a carboxyl group-activated? -Polyglutamic acid derivative and a polyethyleneglycol-based polymer having a plurality of nucleophilic functional groups is contained in the body at a temperature ranging from 25 to 40 占 폚, for example, And gelation may occur within 10 minutes, particularly within 2 minutes, more specifically within 5 to 30 seconds, upon mixing to form a hydrogel. Therefore, such a composition can be gelled and adhered within a short period of time after it is applied to a living body tissue, so that it can be preferably applied in a medical field.

The method of applying the above-described tissue adhesive composition is the same as that already described for the kit for producing a hydrogel, and a further explanation thereof will be omitted.

The tissue adhesive composition can be applied to various applications such as local wound suturing, gastro-intestinal anastomosis, vascular anastomosis, ophthalmic surgery, and the like.

The? -Polyglutamic acid derivatives according to the present invention and the hydrogels produced therefrom maintain the activity for a certain period of time in the aqueous solution state during the surgical operation, so that the solution is not required to be prepared and used immediately. The gel is formed immediately from the wound site, and the adhesive force to the biotissue is excellent, so that leakage of blood or air is suppressed, and it is decomposed and absorbed or discharged in the living body, and there is no toxicity to the living body. The gelation time can also be adjusted and controlled to a desired degree.

Therefore, the hydrogel can be used preferably for various purposes such as biotissue bonding and the like.

Fig. 1 is a graph showing the stability of an activated? -Polyglutamic acid derivative in an aqueous solution through a change in rupture strength of the gel over time after dissolution.
FIG. 2 is a view showing the degradation degree of a hydrogel prepared by cross-linking an activated γ-polyglutamic acid derivative with polyethylene glycol.
FIG. 3 is a ¹ H NMR spectrum showing the content of substituted NHS in activated SS-PGA according to Experimental Example 1 of the present invention.
4 is a ¹ H NMR spectrum showing the content of substituted NHS in activated SG-PGA according to Experimental Example 1 of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

Manufacturing example  1. Activated SS - PGA Manufacturing

Manufacturing example  1.1. γ- PGA  Lt; RTI ID = 0.0 > gamma- PGA - MEA Manufacturing

100 mmol (12.9 g) of γ-polyglutamic acid (PGA, molecular weight 50K, 500K, 1000K, 2000K Da) based on the carboxyl unit was added to a dried 1000 mL round two-necked glass flask, and dimethylsulfoxide (DMSO) And the mixture was stirred at 60 ° C for 16 hours to dissolve uniformly, and then the temperature of the reaction solution was lowered to room temperature (25 ° C). (NHS) and dicyclohexylcarbodiimide (DCC) were each quantitatively determined in excess of 2 equivalents based on the carboxyl unit of? -Polyglutamic acid, sodium bicarbonate was mixed with? -Polyglutamic acid The mixture was weighed in the same molar ratio, and water was removed while stirring under reduced pressure. After one hour, they were reacted under nitrogen for 3 hours. After completion of the reaction, monoethanolamine (MEA) was quantitatively determined in an excess amount of 2 equivalents based on the carboxyl unit of? -Polyglutamic acid, and the mixture was reacted for 1 hour.

After completion of the reaction, the reaction solution was filtrated and precipitated in 4 L of ethyl acetate (EA) at the same time to remove sodium hydrogencarbonate and generated urea. EA to remove unreacted NHS and DCC, and then dried in a vacuum oven for over 16 hours to remove residual solvent. Finally, a linker-introduced? -Polyglutamic acid (? PGA-MEA) compound was obtained.

Manufacturing example  1.2. When an alkyl group is introduced at the linker terminal Carboxy group  S-PGA (Succinylated PGA )

100 mmol (18.3 g) of the γ-PGA-MEA prepared in Preparation 1.1 was added to a dried 1000 ml round-bottomed two-necked glass flask and 450 ml of dimethylsulfoxide (DMSO) 25 ° C) for 2 hours to dissolve uniformly. Succinic anhydride (SA) was quantitatively determined to 6 equivalents over the hydroxyl unit of? -PGA-MEA, and sodium bicarbonate was added at 1 / 2 level, and water was removed by stirring under reduced pressure. After one hour, they were reacted under nitrogen for 24 hours.

After completion of the reaction, the reaction solution was filtrated and precipitated in 2.7 L of ethyl acetate (EA) to remove sodium hydrogen carbonate. EA to completely remove unreacted SA, and then dried in a vacuum oven for 16 hours or more to remove residual solvent.

Manufacturing example  1.3. Active SS - PGA ( Succinimidyl succinyl PGA )

100 mmol (27.28 g) of S-PGA prepared in Preparation Example 1.2 was added to a dried 1000 ml round-bottomed two-necked glass flask, and 700 ml of dimethylsulfoxide (DMSO) (NHS) and dicyclohexylcarbodiimide (DCC) were added to the molar unit of the carboxyl group formed through the reaction with the SA, respectively, Were added in quantitative amounts according to the molar ratios shown in Table 1, and water was removed while stirring under reduced pressure. And reacted for 24 hours under nitrogen for one hour.

After the completion of the reaction, the reaction solution was filtered and simultaneously precipitated in 5.6 L of ethyl acetate (EA) to remove the generated urea. EA to remove unreacted NHS and DCC, and then dried in a vacuum oven for 3 hours to remove residual solvent, then heated to 60 ° C and dried for 72 hours. Finally, an activated SS-PGA ester compound was obtained.

Manufacturing example  2. Activated SG - PGA Manufacturing

Manufacturing example  2.1. G- PGA ( Glutarylated PGA )

100 mmol (18.3 g) of the γ-PGA-MEA prepared in Preparation 1.1 was added to a dried 1000 ml round-bottomed two-necked glass flask and 450 ml of dimethylsulfoxide (DMSO) 25 ° C) for 2 hours to dissolve uniformly. Then, glutaric anhydride (GA) was quantitatively determined to 6 equivalents over hydroxyl unit of? -PGA-MEA and sodium bicarbonate was added at 1 / 2 level, and water was removed by stirring under reduced pressure. After one hour, they were reacted under nitrogen for 24 hours.

After completion of the reaction, the reaction solution was filtrated and precipitated in 2.7 L of ethyl acetate (EA) to remove sodium hydrogen carbonate. EA to remove unreacted GA completely and then dried in a vacuum oven for 16 hours or more to remove residual solvent.

Manufacturing example  2.2. Active SG - PGA ( Succinimidyl glutaryl PGA )

100 mmol (27.28 g) of the G-PGA prepared in Preparation Example 2.1 was added to a dried 1000 ml round-bottomed two-necked glass flask, and 700 ml of dimethylsulfoxide (DMSO) (NHS) and dicyclohexylcarbodiimide (DCC) were added to the molar unit of the carboxyl group formed through the reaction with the GA, respectively, 1, and water was removed by stirring under reduced pressure. And reacted for 24 hours under nitrogen for one hour.

After the completion of the reaction, the reaction solution was filtered and simultaneously precipitated in 5.6 L of ethyl acetate (EA) to remove the generated urea. EA to remove unreacted NHS and DCC, and then dried in a vacuum oven for 3 hours to remove residual solvent, then heated to 60 ° C and dried for 72 hours. Finally, an activated SG-PGA ester compound was obtained.

Experimental Example  1. substituted NHS The content of

The contents of the activated SS-PGA and the substituted NHS in the activated SG-PGA prepared according to Preparation Examples 1 and 2 were measured by NMR to be shown in Table 1 and 3 (SS-PGA) and 4 (SG -PGA). Specifically, the ratio of the integral value of -CH ₂ - (2.8 ppm) of the combined N-hydroxysuccinimide (NHS) to the integral value of -CH ₂ - (2.05 ppm) of γ-PGA measured in 1H NMR (D2O solvent) Respectively.

NHS introduction rate of γ-PGA with NHS and DCC input number ? -PGA weight average molecular weight (kDa) [NHS] / [COOH] [DCC] / [COOH] Substituted NHS content (mol%) T-1 50 2.0 2.0 65 T-2 1,000 1.0 1.0 35 T-3 1,000 1.5 1.5 53 T-4 1,000 2.0 2.0 64 T-5 1,000 3.0 3.0 81 T-6 1,000 4.0 4.0 84

Referring to Table 1, when the reaction was carried out at room temperature for 24 hours, the degree of substitution of NHS increased as the amount of NHS and DCC increased regardless of the γ-PGA weight average molecular weight. However, even when NHS and DCC were reacted in excess of 3 equivalents with respect to the carboxyl group, only 80 mol% of the entire carboxyl groups were substituted.

Example  1 to 6. Activated γ- Polyglutamic acid  Derivatives and Polyethylene glycol series  By concentration of polymer Hydrogels  Produce

The activated γ-polyglutamic acid derivative (SS-PGA) synthesized in accordance with the T-1 conditions in Table 1 and the 4-arm PEG-SH having a molecular weight of 20 kDa were crosslinked (Examples 1 to 3). SS-PGA synthesized in accordance with T-4 conditions in Table 1 and 4-arm PEG-SH having a molecular weight of 20 kDa were crosslinked (Examples 4 to 6).

An SS-PGA aqueous solution (first solution) was prepared by dissolving SS-PGA in 1 ml of an aqueous solution of 0.05 M sodium phosphate in the two tables shown in Table 2. Similarly, a 4-arm PEG-SH aqueous solution (second solution) was prepared by dissolving 4-arm PEG-SH in 1 ml of a 0.3 M sodium phosphate / sodium carbonate mixed solution (5: 5) 0.5 ml of each of the first and second solutions was collected into a 1 ml syringe. Two syringes were mounted on a dual barrel syringe and primed in spray.

Cross-linking reaction conditions of SS-PGA (buffer: 0.05 M sodium phosphate aqueous solution) and polyethylene glycol type polymer (buffer: 0.3 M P / C aqueous solution) number The weight average molecular weight (kDa) of? SS-PGA
(First solution)
(%) Polyethylene glycol-based polymer Polyethylene glycol-based polymer
(%) Of the second solution (second solution) Kinds Molecular Weight
(kDa) Example 1 50 7 4-PEG-SH 20 12 Example 2 50 7 4-PEG-SH 20 10 Example 3 50 6 4-PEG-SH 20 10 Example 4 1000 8 4-PEG-SH 20 12 Example 5 1000 10 4-PEG-SH 20 10 Example 6 1000 12 4-PEG-SH 20 10

Example  7 to 16. Polyethylene glycol series  Types of Polymers Hydrogels  Produce

The activated γ-polyglutamic acid derivative (SS-PGA) synthesized according to the T-4 conditions in Table 1 and the polyethylene glycol-based polymer were cross-linked. An SS-PGA aqueous solution (first solution) was prepared by dissolving SS-PGA in 1 ml of an aqueous 0.05 M sodium phosphate solution in the amounts shown in Table 3. Similarly, an aqueous solution (second solution) was prepared by dissolving the polyethylene glycol-based polymer in 1 ml of a 0.3 M aqueous solution of sodium phosphate / sodium carbonate in the amounts shown in Table 3. 0.5 ml of each of the first solution and the second solution was collected into a 1 ml syringe. Two syringes were mounted on a dual barrel syringe and primed in spray.

Cross-linking reaction conditions of SS-PGA (buffer: 0.05 M sodium phosphate aqueous solution) and polyethylene glycol type polymer (buffer: 0.3 M P / C aqueous solution) number The weight average molecular weight (kDa) of? SS-PGA
(First solution)
(%) Polyethylene glycol-based polymer Polyethylene glycol-based polymer
(%) Of the second solution (second solution) Kinds Molecular Weight
(kDa) Example 7 1000 10 _2-PEG-NH 2 10 10 Example 8 1000 10 _2-PEG-NH 2 20 10 Example 9 1000 10 4-PEG-NH ₂ 20 10 Example 10 1000 12 4-PEG-NH ₂ 20 10 Example 11 1000 12 4-PEG-SH 10 6 Example 12 1000 12 4-PEG-SH 10 One Example 13 1000 12 6-PEG-SH 10 2 Example 14 1000 10 6-PEG-SH 10 2 Example 15 1000 12 6-PEG-SH 20 4 Example 16 1000 12 6-PEG-SH 20 8

Test Example  2. Gelling time  Measure

0.5 ml of each of the first reaction solution and the second reaction solution prepared in the above Examples 1 to 16 was taken in a 1 ml syringe, and then the two liquids were stirred in a 24-well cell culture plate of transparent polystyrene using a magnetic stirrer Respectively. The mixture was stirred at a speed of 500 rpm at room temperature using a magnetic stirrer having a diameter of 4 mm and a length of 12 mm. The time from the start of the first reaction solution and the second reaction solution to the stop of the magnetic stirrer was measured with a stopwatch. The results are shown in Table 4.

Gelation time of SS-PGA and polyethylene glycol derivative number Gel time
(second) Example 1 5 Example 2 7 Example 3 7 Example 4 8 Example 5 5 Example 6 2 Example 7 N / G Example 8 N / G Example 9 4 Example 10 5 Example 11 4 Example 12 30 Example 13 25 Example 14 21 Example 15 10 Example 16 One

In the cross-linking reaction between the activated γ-polyglutamic acid derivative and the polyethylene glycol-based polymer, the degree of substitution of NHS is similar regardless of the molecular weight of γ-polyglutamic acid, and the gelation time does not show any significant difference. Under the same conditions, the gelation time decreased with increasing the total polymer concentration. However, when the concentration of the polymer was more than 10%, the gel was partially formed due to the gelation which was too fast, so that a uniform gel could not be formed.

On the other hand, in Example 7 and Example 8 where 2-PEG-NH2 was used as the polyethylene glycol-based polymer, no gelation reaction occurred regardless of the molecular weight of PEG. In Examples 13 to 16 in which crosslinking reaction with 6-arm PEG-SH was performed, fast gelation occurred at a relatively lower concentration because the number of reactors per unit of PEG was larger than that of 4-arm PEG-SH. However, as the concentration of 6-arm PEG-SH increased, gels formed non-uniformly due to too fast gelation.

Test Example  3. Rupture strength  Measure

The rupture strength was measured by the method specified in ASTM2392. 0.35 ml of each of the first reaction solution and the second reaction solution prepared in Examples 1 to 16 was taken in a 1 ml syringe. The collagen casing was washed twice with water and ethanol to remove glycerin from the collagen casing, and then cut to a size of 3 × 3 cm. Using a punch for skin biopsy, a 3 mm hole was drilled and used as a tissue substitute. Thereafter, a collagen casing having a 3 mm hole was fixed with Teflon as a support. Thereafter, the first reaction solution and the second reaction solution were mixed with 0.3 ml each using a dual barrel syringe, and the reaction solution was applied to the hole of the collagen casing so that the volume of the reaction solution became 0.6 ml, followed by allowing to stand for 5 minutes to cure. Thereafter, the collagen casing coated with the mixed reaction liquid is separated from the Teflon support, fixed on the burst strength meter manufactured by the method specified in ASTM2392, and the water pressure is measured. The cured gel breaks and the measured water pressure is taken as the burst strength. Table 5 shows the rupture strength results of the hydrogels.

Rupture Strength of SS-PGA and Polyethylene Glycol Derivatives number Burst strength
(mmHg) Example 1 191.5 Example 2 180 Example 3 165 Example 4 105 Example 5 180 Example 6 137 Example 7 N / G Example 8 N / G Example 9 240 Example 10 220 Example 11 118 Example 12 58 Example 13 80 Example 14 84 Example 15 148 Example 16 30

A. γ- Polyglutamic acid  Effect of Polymer Concentration

The maintenance of the tear strength above a certain level is influenced by the concentration of the activated γ-polyglutamic acid polymer. Comparing Examples 2, 3, 5, and 6 using the same 4-arm PEG-SH polymer concentration, it was found that as the concentration of γ-PGA polymer increases, the tear strength also increases. However, when the concentration of γ-PGA polymer was more than 10%, the tear strength did not increase any more, and the tear strength decreased at 12%. This is because the concentration of the polymer was too high to lower the solubility and the viscosity could not be increased so that a uniform gel could not be formed during the mixing of the first liquid and the second liquid.

Also, when the total polymer concentration was 9 to 10%, the tear strength was measured to be high.

Even if the concentration of 4-arm PEG-SH polymer is increased to 10% of the total polymer concentration, if the concentration of activated γ-polyglutamic acid polymer falls below 6% for molecular weight 50k and below 8% for 1000k, (See Examples 3 and 4).

B. Polyethylene glycol series  Effect of Polymer Concentration

Comparing Examples 1 and 2 with Examples 11 and 12, in the case of a hydrogel prepared by varying the concentration of the polyethylene glycol-based polymer under the same conditions, the bursting strength also increases as the concentration of the polyethylene glycol-derived polymer increases I could confirm.

When 6-PEG-SH was used as a polyethylene glycol-based polymer, a uniform gel was formed even at a lower concentration because the number of reactors per unit of PEG was larger than that of 4-arm PEG-SH. However, A lower tear strength value was measured than the gelling reaction with -arm PEG-SH. When the concentration of 6-arm PEG-SH was too high (Example 16), the gel was unevenly formed due to gelation too fast, which was just one second, and very low tear strength values were measured. Conversely, if the concentration was too low The gel was weakly formed due to lack of crosslinking points.

C. Influence of Polymer Types

Example 5 in which the concentration of γ-PGA and polyethylene glycol-based polymer and the molecular weight of γ-PGA were the same and only the types of polyethylene glycol-based polymers were different from 4-PEG-SH and 4-PEG-NH ₂ , respectively 9 and Examples 6 and 10 showed higher rupture strengths compared to Examples 5 and 6 using 4-PEG-SH in Examples 9 and 10 prepared using 4-PEG-NH ₂ , In addition, the hydrogel according to Examples 1 to 16 showed the highest rupture strength.

However, in Examples 6 and 10 in which the concentration of? -PGA was 12%, the rupture strength was lower than that in Examples 5 and 9 in which the concentration of? -PGA was 10% . This is because the concentration of the polymer was too high to lower the solubility and the viscosity could not be increased so that a uniform gel could not be formed during the mixing of the first liquid and the second liquid.

On the other hand, in the case of Examples 7 and 8 prepared using 2-PEG-NH ₂ , as mentioned in the measurement of the gelation time in Test Example 2, no gelling reaction occurred and the bursting strength could not be measured.

Test Example  4. Measurement of adhesion strength

0.15 ml of each of the first reaction solution and the second reaction solution prepared in accordance with the Example was taken in a 1 ml syringe. The frozen pig skin was degassed at room temperature and then degreased and cut to 1 × 5 cm. In addition, the collagen casing was washed twice with water and ethanol, respectively, and cut to 1 × 5 cm. The first reaction solution and the second reaction solution collected in a 1 ml syringe were mixed with 0.1 ml each using a dual barrel syringe so that the volume of the reaction solution became 0.2 ml and the surface of the pig skin or the collagen casing was immersed in a solution of 1 × 1 cm ² ). After attaching pig skin or collagen casing of the same size, a load of 50 g was applied and allowed to stand for 10 minutes to cure the gel. After 10 minutes, the load was removed and shear force was continuously applied at a rate of 100 mm / min until the bonded pig skin or collagen casing was peeled from each other by using a tensile tester (H5K-T, Hounsfield) The load was taken as the adhesive strength. The same test was also performed for fibrin glue (Beriplast ?, CSL Behring) for comparison. The results of adhesion strength of the hydrogel are shown in Table 6.

Adhesion strength between SS-PGA and polyethylene glycol derivatives No. Adhesion strength (gf / cm ² ) Example 5 398 Example 6 148 Example 10 238 Example 14 65 Example 15 196

It was confirmed that the adhesive strength was similar to the burst strength. When the gelation time was about 5 seconds, gelation occurred between the two materials, and at the same time, the bond strength with 400 gf / cm was measured. However, when the gelation time was too fast, the bonding strength was measured to be low because there was not enough time to bond to collagen caking.

Test Example  5. Stability in aqueous solutions ( pot life ) test

The stability of SS-PGA in aqueous solution was determined by measuring the burst strength over time after dissolution. The rupture strength was measured by the method specified in ASTM2392. After the aqueous solution of SS-PGA aqueous solution (first reaction solution) and the aqueous solution of polyethylene glycol derivative (second reaction solution) were prepared in the same manner as in Examples 1 and 5, 0.35 ml of each aqueous solution was taken in each 1 ml syringe. The collagen casing was washed twice with water and ethanol, and the glycerin in the collagen casing was removed. Then, the collagen casing was cut to 3 × 3 cm, and a 3 mm hole was drilled using a skin biopsy punch. Thereafter, a collagen casing having a 3 mm hole was fixed with Teflon as a support. The first reaction solution and the second reaction solution were mixed with 0.3 ml each using a double barrel syringe to make the volume of the reaction solution to be 0.6 ml, and the mixture was applied to the hole of the collagen casing, and allowed to stand for 5 minutes for curing. Thereafter, the collagen casing coated with the mixed reaction solution was separated from the Teflon support, fixed on the rupture strength meter manufactured by the method specified in ASTM2392, and then the water pressure was measured.

The water pressure measured as the cured gel was broken was taken as the tear strength. The change was measured for 20 hours at intervals of 20 minutes to 30 minutes immediately after the first reaction solution was prepared, and the stability of the hydro- The stability results of the gel in an aqueous solution are shown in Fig.

As shown in FIG. 1, the stability of an aqueous solution of hydrogels according to Examples 1 and 5 was evaluated using a hydrogel prepared using? -Polyglutamic acid activated by a conventional succinimide group (Korean Patent Application No. 10- ([NHS] / [COOH] = 1.2, [DCC] / [COOH] = 1.5, reaction time 3 hours) prepared in accordance with Production Example T- The NHS was rapidly deactivated in γ-polyglutamic acid activated by the existing succinimide group in the aqueous solution, and the bursting strength was reduced to about 30 mmHg within 40 minutes, resulting in a 20% level of initial (150 mmHg) While the initial physical properties were maintained when the hydrogel of Example 1 was measured up to 2 hours and the initial physical properties were maintained up to about 1 hour after the hydrogel of Example 5. However, Number of The gel was found to fall to 50% of the initial strength after 1 hour, since the concentration of activated? -Polyglutamic acid (10%) of Example 5 is higher than the concentration of 7% of Example 1 The amount of free NHS in the aqueous solution became larger and the stability was deteriorated, so that the value of the tear strength according to time was measured to be lower.

Test Example  6. Exploded view ( in vitro degradation ) test

To investigate the hydrolysis behavior of hydrogels, 1 g of the hydrogel prepared according to Example 5 was placed in 50 ml of PBS and immersed in a thermostatic chamber at 37 ° C and 47 ° C, 50 rpm for a predetermined period (1 to 5 weeks, 1 week interval) The results are shown in Fig. The weight change by hydrolysis of hydrogel was measured by freeze drying and weight ratio before and after decomposition.

As shown in FIG. 2 showing the result of weight loss due to the hydrolysis of the hydrogels, when the PBS temperature was 47 ° C, the hydrolyzate rapidly disintegrated within one week after one week, Was gradually decomposed after 1 week at 37 ℃, remaining at 10% of initial weight at 4 weeks and completely degraded at 5 weeks. As described above, it was confirmed that the hydrogel according to one preferred embodiment of the present invention exhibits favorable decomposition behavior since decomposition starts within one week after completion of wound healing in the body and is completely decomposed within two months.

Claims (12)

A? -Polyglutamic acid derivative represented by the following formula 1:
[Chemical Formula 1]

In Formula 1,
The sum of l, m and n is an integer from 390 to 15,500,
Wherein the ratio of 1, m and n is 1: m: n = 0 to 0.5: 0.2 to 0.5: 0.2 to 0.8,
L is a linker, M is independently H, an alkali metal or an alkaline earth metal, R is CH ₂ ,
b is 0 or 1, and c is an integer of 1 to 5.
The method according to claim 1,
Wherein the linker is -HN- (R) aO- (wherein R is CH ₂ and a is an integer of 1 to 5).
3. The method of claim 2,
The linker may be selected from the group consisting of aminomethanol, 1-amino-2-propanol, 1-amino-3-propanol, Those derived from 1-amino-4-butanol, 1-amino-5-pentanol or 1-amino-2-ethanol (MEA or 1-amino-2- Phosphoric acid derivatives.
According to claim 1, - (CO) b- (R ) c-CO- is _{-CH 2 CH 2 CH 2 CH 2} -CO-, -CO-CH 2 CH 2 CH 2 -CO-, -CH 2 CH ₂ -CO-, -CO-CH ₂ CH ₂ -CO- or -CH ₂ -CO-.
a first step of reacting? -polyglutamic acid with a lower alkanolamine having 1 to 5 carbon atoms to form? -polyglutamic acid-alkanolamine;
Wherein the? -Polyglutamic acid-alkanolamine is reacted with an anhydride of an acid selected from the group consisting of glutaric acid and succinic acid, or an anhydride of an acid selected from the group consisting of 1-halovaleric acid, 1-halopropionic acid and 1-halomethylcarbonic acid A second step of reacting with 1-halo alkanoic acid to form a carboxy terminal introduced with an alkyl group; And
A third step of reacting the formed carboxyl terminal with N-hydroxysuccinimide or N-hydroxysulfosuccinimide to form at least a part of the carboxyl group-activated? -Polyglutamic acid
Polyglutamic acid derivative according to any one of claims 1 to 4,
6. The method of claim 5, wherein step
Wherein the lower alkanolamine is reacted at a molar ratio of 0.1 to 2 to the molar unit of the carboxyl group of? -Polyglutamic acid.
6. The method of claim 5, wherein step
Wherein the anhydride of the acid or the 1-haloalkanoic acid is reacted in a molar ratio of 3 to 8 with respect to the molar unit of the lower alkanolamine forming the? -Polyglutamic acid-alkanolamine, wherein the? -Polyglutamic acid derivative Way.
6. The method of claim 5, wherein step
Wherein the N-hydroxysuccinimide or N-hydroxysulfosuccinimide is reacted at a molar ratio of 0.1 to 3 to the molar unit of the formed carboxy terminal.
A method for producing a γ-polyglutamic acid derivative according to any one of claims 1 to 4,
Wherein the polyethylene glycol-based polymer having a plurality of nucleophilic functional groups is crosslinked.
10. The method of claim 9,
Wherein the nucleophilic functional group is an amine group, a thiol group or a hydroxy group.
10. The method of claim 9,
The polyethylene glycol-based polymer may be a hydrogel of formula (2)
(2)

Wherein X is an amine group, a thiol group or a hydroxy group, n is from 19 to 170, m is an integer from 2 to 12, and I is a group derived from a polyhydric alcohol The number of hydroxyl groups in the resulting polyhydric alcohol.
12. The method of claim 11,
The polyethylene glycol-based polymer may be a hydrogel of the following formula (3) or (4)
(3)

[Chemical Formula 4]

In the above formulas (3) and (4), X represents an amine group, a thiol group or a hydroxyl group, and n is 19 to 170.

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